tau Exon 10 Expression Involves a Bipartite Intron 10 RegulatorySequence and Weak 5� and 3� Splice Sites*

Received for publication, April 19, 2002Published, JBC Papers in Press, May 8, 2002, DOI 10.1074/jbc.M203794200

Ian D’Souza‡§ and Gerard D. Schellenberg‡§¶�

From the ‡Divisions of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington,Seattle, Washington 98195, the §Geriatric Research Education and Clinical Center, Veterans Affairs Puget Sound HealthCare System, Seattle Division, Seattle, Washington 98108, and the ¶Departments of Pharmacology and Neurology,University of Washington, Seattle, Washington 98195

tau mutations that deregulate alternative exon 10(E10) splicing cause frontotemporal dementia with par-kinsonism chromosome 17-type by several mechanisms.Previously we showed that E10 splicing involved exonsplicing enhancer sequences at the 5� and 3� ends of E10,an exon splicing silencer, a weak 5� splice site, and anintron splicing silencer (ISS) within intron 10 (I10).Here, we identify additional regulatory sequences in I10using both non-neuronal and neuronal cells. The ISSsequence extends from I10 nucleotides 11–18, which issufficient to inhibit use of a weakened 5� splice site of aheterologous exon. Furthermore, ISS function is loca-tion-independent but requires proximity to a weak 5�splice site. Thus, the ISS functions as a linear sequence.A new cis-acting element, the intron splicing modulator(ISM), was identified immediately downstream of theISS at I10 positions 19–26. The ISM and ISS form a bi-partite regulatory element, within which the ISM func-tions when the ISS is present, mitigating E10 repressionby the ISS. Additionally, the 3� splice site of E10 is weakand requires exon splicing enhancer elements for effi-cient E10 inclusion. Thus far, tau FTDP-17 splicing mu-tations affect six predicted cis-regulatory sequences.

The tau protein is a microtubule (MT)1-associated proteininvolved in MT assembly, stability, and function (1, 2). It islocalized in neuronal axons and may be involved in MT-medi-ated axonal transport. MAPT, the gene encoding tau, has 15exons, and transcripts of the gene are alternatively spliced toproduce different isoforms (3). Isoform expression patterns areunder developmental control (4, 5). In fetal human brain, asingle tau isoform is expressed containing exons 1, 4, 5, 7, 9,and 11–13. In adult human brain, five additional variants areexpressed by alternative splicing of exons 2, 3, and 10. Theconditional inclusion of exon 10 (E10), which encodes a MT-

binding domain, generates tau isoforms with four MT-bindingrepeats (4R tau) compared with isoforms without E10 thathave only three MT-binding repeats (3R tau).

The neuropathological aggregation of tau as neurofibrillarytangles occurs in several progressive dementias includingAlzheimer’s disease, Pick’s disease, corticobasal degenera-tion, progressive supranuclear palsy, and frontotemporal de-mentia with parkinsonism chromosome 17-type (FTDP-17) (6).FTDP-17 represents a phenotypically heterogeneous group ofinherited dementias caused by mutations in MAPT (7–9). Twoclasses of tau mutations are defined by in vitro functionalanalyses. One class, represented by mutations G272V (E9),P301L, P301S, and �280K (E10), V337M (E12), and R406W(E13), results in tau with altered biochemical properties. Thesemutations affect tau-MT interaction properties, reducing theaffinity of tau for MT and/or the ability of tau to stimulate MTpolymerization (10–14). Some of these mutations (P301L,�280K) also alter tau self-aggregation properties, acceleratingthe in vitro formation of filaments that resemble filamentsfrom neurofibrillary tangle-containing disease brains (10, 15).

A second class of mutations alters the relative levels of 4Rversus 3R tau by affecting alternative splicing of E10 (7, 9, 11,16–20). These include missense (N279K, N296H, and S305N),silent (L284L, N296N), and deletion mutations (�280K,�296N) in E10, and intronic mutations (E10�3, E10�11,E10�12, E10�13, E10�14, and E10�16; Fig. 1A) in intron 10(I10). Most of these mutations increase E10 inclusion. Theresult is that the 4R/3R ratio, which is normally 1 (14), isincreased to 2–3 (7, 16). One mutation, �280K, reduces E10inclusion to an E10�/E10� ratio of 0.33. Thus, subtle alter-ations in the 4R/3R ratio lead to FTDP-17, a severe neurode-generative disease.

The FTDP-17 splicing mutations in E10 and I10 indicatethat multiple cis-acting regulatory elements control E10 inclu-sion. Previous work demonstrated that E10 splicing is complex,involving exonic and intronic regulatory elements that interactto modulate use of the weak E10 5� splice site (21). The exonicelements include three nonredundant exon splicing enhancer(ESE) sequences located within the first 45 bases of E10. Theseare a 5� SC35-like sequence, a polypurine enhancer (PPE), anda 3� A/C-rich enhancer (ACE). Additional E10 elements includean exon splicing silencer (ESS) and two adjacent 9-nucleotidepositive sequences immediately upstream of the 5� splice site.The I10 intronic regulatory element is the intron splicing si-lencer (ISS) located downstream of the 5� splice. FTDP-17mutations affect six of these cis-acting regulatory sequences(see “Discussion”).

In previous work, multiple cis-acting E10 regulatory ele-ments were identified that interact with the weak 5� splice siteand the ISS (11, 21). Here, the role of flanking intronic se-

* This work was supported by Grant RO1 AG11762 from the NIA,National Institutes of Health and by a grant from the Department ofVeterans Affairs (to G. D. S.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

� To whom correspondence should be addressed. Tel.: 206-764-2701;E-mail: zachdad@u.washington.edu.

1 The abbreviations used are: MT, microtubule; ACE, A/C-rich en-hancer; BPS, branch point sequence; E10, exon 10; ESE, exon splicingenhancer; ESS, exon splicing silencer; FTDP-17, frontotemporal demen-tia with parkinsonism chromosome 17-type; I9, intron 9; I10, intron 10;ISM, intron splicing modulator; ISS, intron splicing silencer; MCS,multiple cloning site; PAC, P1 artificial chromosome; PPE, polypurineenhancer; PPT, polypyrimidine tract; snRNP, small nuclear ribonucle-oprotein; SR, Arg/Ser-rich splicing factors; 3R, 3-repeat tau; 4R, 4-re-peat tau; �E2, �-globin exon 2; RT, reverse transcription; HIV, humanimmunodeficiency virus.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 29, Issue of July 19, pp. 26587–26599, 2002Printed in U.S.A.

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quences on E10 splicing regulation is examined. The resultsshow that both I9 and I10 sequences regulate E10 inclusion. InI10, a novel bipartite regulatory element composed of the in-hibitory ISS and an adjacent intron splicing modulator (ISM)sequence affect E10 inclusion. The data are consistent with theISS functioning as a linear element rather than through asecondary structure as previously proposed (7, 9). The E10 3�splice site, like the 5� splice site, is also weak and its activity isdependent on E10 ESEs. Thus, there is a complex interplaybetween I9, E10, and I10 regulatory sequences that maintainsthe normal ratio of 4R versus 3R tau. FTDP-17 mutationsdisrupt this complex regulatory system by multiple mecha-nisms leading to distinct disease phenotypes.

EXPERIMENTAL PROCEDURES

Plasmid Construction and DNA Mutagenesis—Splicing was assayedusing a derivative of vector pSPL3 called pSPES (21). pSPL3 containsan HIV genomic fragment with a multiple cloning site (MCS) betweenhighly truncated tat exons 2 and 3 fused to rabbit �-globin codingsequences (22). pSPES was generated from pSPL3 by deleting a 500-bpfragment between the EcoRV (nucleotide 1050) and StuI (nucleotide1550) sites, removing a previously reported cryptic exon within the tatintron. Construct hN contains human tau E10 with 33 and 51 bp offlanking introns inserted into the MCS of pSPES as previously de-scribed (21). In hN and other similar tau constructs, the weak E10 5�splice site activates a 60-bp cryptic exon between the MCS and thedownstream tat/globin hybrid exon 3. Modifications within tau se-quences were performed by PCR mutagenesis (11, 21). ConstructE10860 is identical to hN but with longer flanking I9 (567 bp) and I10(190 bp) sequences. The E10860 insert was generated by PCR from PACclone 4139 using forward primer I9AF (5�-GGACTAGTGAGACT-GAAGCCAGACTCCTAGATT-3�) and reverse primer I10AR (5�-ATG-CATCCTCACACTGGGAACAGTGGACCATG-3�). The PCR productwas blunt-end ligated into the MCS of pSPES.

Human �-globin exon 2 (�E2) with 42 and 14 bp of flanking intronswas PCR amplified from PAC P�(BglII) using forward and reverseprimers G1XhoF (5�-CCGCTCGAGCACTGACTCTCTCTGCCTATTGG-3�) and GE2BamR (5�-CGGGATCCCATAGACTCACCCTGAAGT-TCTCACGATCC-3�), respectively. The PCR product was digested withXhoI/BamHI and inserted into the pSPES MCS to generate GE2. Forcloning purposes, the BamHI site in �E2 was destroyed and a syntheticBamHI site introduced into intron 2 by incorporating point mutations(G to C) and (c to t) at �E2 position �14 and intron 2 position �15,respectively. Mutant GE2/ISS contains the ISS sequence 5�-tcacacgt-3�inserted 10 bp downstream of �E2 using mutagenized primer GE2/ISS(5�-CGGGATCCACGTGTGAATAGACTCACCCTGAAGTTCTCACGA-TCC-3�) with G1XF. In mutant GE2/ISS�14 the ISS sequence was neu-tralized to 5�-tcatacgt-3� by a point mutation (underlined). All sequenceswere confirmed by dye terminator cycle sequencing with BigDye(PerkinElmer Life Sciences) using an ABI377 DNA sequencer.

Cell Culture, Transfection, and RNA Isolation—COS-7 cells werecultured and transfected as previously described (11). PC12 cells weremaintained in Dulbecco’s modified Eagle’s medium supplemented with10% horse serum and 5% fetal bovine serum (Invitrogen) and seededonto poly-D-lysine-coated six-well plates to achieve 70% confluence 1day prior to transfection. Primary cortical neurons were isolated frompost-natal day 1 Sprague-Dawley rat pups cultured for 5–7 days prior totransfection as previously described (23). PC12 cells (5 � 105) weretransfected using 2 �g of plasmid DNA and 10 �l of LipofectAMINE in1 ml of Opti-MEM (Invitrogen) for 5 h at 37 °C (5% CO2), after which 1ml of Dulbecco’s modified Eagle’s medium supplemented with 20%horse serum and 10% fetal bovine serum was added. Primary corticalneurons were transfected with 1 �g of plasmid DNA mixed with 10 �lof the polycationic liposomal reagent DOSPER (Roche) and incubated atroom temperature for 30–45 min in a total of 60 �l of HEPES-bufferedsaline (137 mM NaCl, 4.8 mM KCl, 0.56 mM Na2PO4, and 21 mM HEPES,pH 7.3). The DNA/DOSPER mixture was added to 1 ml of Neurobasalmedium (Invitrogen) containing 0.32 mM glutamine. Culture mediumwas removed and stored before adding the DNA/DOSPER/Neurobasalmixture (23). After 5 h, conditioned medium was added back to allowneurons to recover. RNA was isolated as previously described (11).

Quantitation of tau E10 Splicing by Reverse Transcription-PCR (RT-PCR)—E10 splicing was assayed by RT-PCR as previously reported (11,21). The E10� and E10� products as amplified by RT-PCR from trans-fected cells are 261 and 354 bp, respectively. For each mutant construct,values presented are the average of at least three different transfection

experiments with the normal tau E10 or �E2 construct also transfectedin parallel. Statistical comparisons were made using a two-tailed Stu-dent’s t test. Criteria for significance were calculated using a Bonferronicorrection for multiple comparisons by dividing the initial p value of0.05 by the number of comparisons made.

RESULTS

Intronic FTDP-17 mutations are clustered in an I10 inhibi-tory element designated the ISS (Fig. 1A). These mutationsdramatically increase E10 inclusion in tau transcripts, demon-strating that I10 sequences regulate E10 inclusion. WhetherI10 sequences downstream of E10�16 also contribute to E10splicing regulation is unknown. Furthermore, the role of I9sequences in E10 inclusion has not been examined. Two mech-anisms for ISS regulation of E10 splicing have been proposed.One is that, normally, the ISS is part of a stem loop (Fig. 1B)that blocks access of U1 snRNP to the 5� splice site, therebyinhibiting use of this splice site and E10 inclusion (7, 9). I10FTDP-17 mutations disrupt base pairing in the stem, resultingin increased availability of the 5� splice site and increased E10inclusion. Three variations of the stem loop that differ in stemlength have been proposed (7, 9, 11), but previous studies showthat only the shortest predicted 18-nucleotide stem loop (Fig.1B) is a candidate for regulating E10 splicing (17, 21, 24). Thesecond hypothesis is that the ISS functions as a linear sequencethat binds trans-acting factors and that FTDP-17 mutations inI10 alter this protein-RNA interaction (11, 21). Here, experi-ments were designed to: 1) define the sequences required forISS function, 2) identify additional I10 regulatory elements, 3)distinguish between the stem loop and linear models, and 4)determine whether I9 contributes to E10 splicing regulation.E10 splicing regulation was evaluated by transfecting COS-7,PC12, and rat primary cortical neuron cultures with vectorpSPES containing E10 and portions of I9 and I10 inserted intoan HIV tat intron between tat exons 2 and 3. E10 splicing wasquantitated by RT-PCR assays of RNA from transfected cells.

I10 Sequences Required for Regulated Splicing—To deter-mine the I10 sequences needed for regulated E10 splicing, aseries of constructs were generated that differ only in theamount of I10 included (6B-37B, Fig. 1A), and cloned into theXhoI/BamHI sites of the MCS of vector pSPES. The FTDP-17mutation E10�3 was incorporated into a second set of con-structs (6B3A to 37B3A) to determine whether the regulationof E10 splicing paralleled that observed in vivo in FTDP-17patients with I10 mutations. The shortest constructs (6B and6B3A) yielded only partial E10 inclusion, probably becausepositive downstream elements are missing (Fig. 1, C and D).Splicing was not influenced by the E10�3 mutation. The 12Band 12B3A constructs showed near-constitutive splicing, pre-sumably because I10 nucleotides 7–12 contain a positive se-quence and the ISS is missing. Construct 18B has reduced E10inclusion, indicating that I10 nucleotides 13–18 contain part orall of the ISS. These sequences restore mutation-sensitive reg-ulated E10 splicing (18B3A). Longer constructs (24B, 37B, andhN) yielded varied amounts of E10 inclusion percentages, in-dicating additional regulatory element(s) exist downstream ofthe ISS.

I10 Has Antagonistic Regulatory Sequences—To further re-fine the locations of the ISS and the positive regulatory se-quences adjacent to the ISS, a series of deletions in I10 wereconstructed, beginning at position 7 and extending to 42 (Fig.1B). Deletion �7–10 removes 4 of 6 nucleotides from the loopsequence. The stem loop hypothesis predicts this deletionwould destabilize the proposed secondary structure, causingE10 to oversplice. However, E10 inclusion was decreased whenthese 4 nucleotides were removed (Fig. 2). Thus, the deletednucleotides contain a positive element, a conclusion that isconsistent with the results with constructs 6B and 12B (Fig. 1).

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Deletions �11–14 and �15–18 allow almost constitutive in-clusion of E10 (Fig. 2). This phenotype is the same as yielded byFTDP-17 point mutations E10�12, E10�13, E10�14, andE10�16 (92, 91, 93, and 89% E10 inclusion, respectively; Ref.21). Deletions �19–22 and �23–26, immediately downstreamof the ISS, severely inhibit E10 inclusion, indicating that apreviously unrecognized positive element is at nucleotides 19–26. Deletion �27–29 shows normal E10 inclusion and definesthe 3� boundary of this positive element. Key deletions �7–10,�12–18, and �19–22 were also tested in primary rat cortical

cells, and the results were comparable with those from COS-7and PC12 cells.

Additional constructs were tested to refine the locations ofthe ISS and the downstream positive element. Deletion �7–11,designed to define the 5� end of the ISS, slightly increased E10inclusion compared with �7–10 (Fig. 2). It appears that ISSfunction is partially compromised in �7–11, which compen-sates for loss of the positive sequence located between positions7 and 10. Thus, the functional ISS sequence begins at I10position 11, a conclusion supported by FTDP-17 mutation

FIG. 1. Deletion analysis of tau I10. A, the 3� end of tau E10 used in construct hN is shown by an open box, the intronic sequence as lowercaseletters, and the 5� splice site as 5�ss. I10 deletion constructs are shown below hN. Each construct was inserted into the BamHI site (underlined)in the MCS of pSPES. BamHI sites were added to the 3� end of each I10 deletion construct to facilitate cloning into pSPES. FTDP-17 mutationsare shown above the sequence. The ISS and ISM regulatory sequences from positions 11–18 and 19–26, respectively, are shown below the I10sequence by solid bars. B, the E10–I10 boundary is shown as a previously proposed stem loop structure (7, 9). Exon nucleotides are in capital lettersand intronic nucleotides in lowercase letters. The locations of deletions and substitutions used in subsequent experiments are shown. C,representative autoradiograph of E10 splicing assays. D, quantitation of E10 splicing assays. Each bar represents the mean of at least threeseparate transfection experiments and 100% is the sum of E10� and E10�. Error bars are standard deviations. A corrected significance criterionof p � 0.003 was used, and p values for comparison of each construct to normal human E10 are indicated with the following symbols: ‡, p � 1 �10�3; �, p � 1 � 10�4; †, p � 1 � 10�5; �, p � 1 � 10�7; �, p � 1 � 10�8. Other comparisons are indicated using lines connecting the bars for theconstructs being compared.

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E10�11A, that causes an increase in E10 splicing (25). Dele-tion �17–18, designed to define the 3� end of the ISS, increasedE10 inclusion. Thus, these 2 nucleotides are part of the ISS.The results from these deletions, from I10 FTDP-17 mutations,and the constructs in Fig. 1 (compare 12B, 18B, and 24B) definethe ISS element as the sequence from 11 to 18. Additionally,these results demonstrate that there is positive element be-tween 19 and 26.

To determine whether the ISS and the positive 19–26 ele-ment interact, the ISS alone or both elements were deleted inmutant �12–18 and �12–24, respectively. Constitutive E10incorporation was observed in both �12–18 and �12–24 (Fig.2). Thus, removal of the ISS relieves inhibition of E10 inclusionregardless of the presence of nucleotides 19–26. Because thepositive element functions only in the presence of an intact ISS(compare deletions �19–22 and �23–26 with �12–18) and ap-pears to counteract ISS function (compare �19–22 and �23–26

with �12–18 and �12–24), it does not behave as a splicingenhancer per se, but rather as a modulator of ISS inhibition ofE10 inclusion. Thus, the E10 19–26 element is designated theintron splicing modulator (ISM). Further evidence supportingthe existence of the ISM is the recent identification of an FTDmutation, E10�19G (Fig. 1A).2 This mutation decreases E10inclusion (Fig. 3), although not as dramatically as removing theentire ISM (�19–26, Fig. 2). It also appears that a purine atposition 19 (mutants E10�19G and E10�19A, Fig. 3) compro-mises ISM function more effectively than a pyrimidine(E10�19T).

Loop Nucleotides 8 and 10 Increase E10 Inclusion—The de-crease in E10 splicing observed in mutant �7–10 indicates this

2 P. M. Stanford, J. B. J. Kwok, and P. R. Schofield, personalcommunication.

FIG. 2. Defining cis-acting sequences in I10. A, representative autoradiographs of E10 splicing assays in COS-7, PC12, and rat P1 neurons.B, quantitation of E10 inclusion. Bar graphs are as in Fig. 1. A corrected significance criterion of p � 0.004 was used for COS-7 and PC12 cells andp � 0.02 for rat primary neurons. Comparisons are for hN to each construct for a given cell type, and p values are as in Fig. 1 and as follows: �,p � 1 � 10�2; *, p � 1 � 10�6.

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sequence is not neutral. Intronic sequences immediately down-stream of the 5� splice site consensus sequence (Fig. 4A) inter-act with splicing machinery components (26, 27). Both U1 andU6 snRNA directly hybridize with the consensus 5� splice sitesequence and these interactions extend to intron position 8 forU1 snRNA and positions 6–10 for U6 snRNA (Fig. 4A; Refs.28–31). Intron position 7 of tau protein is an A that is compat-ible with the corresponding U nucleotide in both U1 and U6snRNAs. However, the C at I10 position 8 is incompatible withthe corresponding A residue in both snRNAs. To increase basepairing between these snRNAs and the tau I10 5� splice site,nucleotides at positions 8 and 10 were individually mutated tocomplementary residues U and G, (mutants c(�8)t andt(�10)g, respectively; Fig. 4). E10 inclusion was enhanced inc(�8)t, but remained unaltered in t(�10)g only in COS-7 cells(Fig. 4). Double mutant c(�8)t/t(�10)g showed a slightly higherincorporation of E10, suggesting that U6 snRNA associationwith I10 is more stable when both I10 positions 8 and 10 arecomplementary. Thus, the E10 5� splice site extends to E10�10and deletion �7–10 weakens 5� splice site interactions withsplicing machinery resulting in reduced E10 inclusion.

The ISS Sequence Can Regulate a Heterologous Exon—Todistinguish between the stem loop and linear models for ISSaction, the 8-nucleotide ISS was inserted 11 nucleotides down-stream of human �-globin exon 2 (�E2) (Fig. 5). �E2 is flankedby 42 and 14 nucleotides of globin intron sequences and in-serted into the MCS of pSPES (GE2, Fig. 5A). If the linearmodel is correct, the ISS should function in this heterologouscontext independent of secondary structure proposed at the tauE10/I10 junction. Although �E2 is a constitutive middle exon, itis included with 76% efficiency in GE2 (Fig. 5, B and C),because of the suboptimal nature of the flanking HIV tat splicesites (32). Previously, we demonstrated that ISS function re-quires a weak 5� splice site (21). Consistent with this previouswork, when the ISS was tested with the normal ��2 5� splice

site in GE2/ISS, the ISS did not alter exon inclusion (Fig. 5, Band C), nor did the addition of FTDP-17 mutation E10�14 inthe ISS (GE2/ISS�14) affect �E2 inclusion. When the �E2 5�splice site was weakened at the �1 position in GE25� (Fig. 5A),making it identical to the tau E10 5� splice site, total levels of�E2 inclusion remained unaltered for GE25� as in GE2 (Fig. 5,B and C). However, 16 and 21% of total E2� transcripts werelarger than expected in COS-7 and PC12 cells, respectively.This larger product, designated �E2a�, was sequenced and theresults showed that the �E2 5� splice site is not used. Rather,an in-frame cryptic 5� splice site 39 nucleotides downstream ofthe �E2 5� splice site is used. Because the total amount of �E2inclusion does not change when GE2 and GE25� are compared,use of a cryptic donor site for �E2a� is at the expense of thenormal 5� splice site. When the ISS sequence was inserted(GE25�/ISS) total �E2� transcript levels did not change (Fig.5C), but the cryptic 5� splice site was greatly favored (89 and93% of �E2� transcripts in COS-7 and PC12 cells, respectively)(Fig. 5D). Compromising the ISS with FTDP-17 mutationE10�14 (GE25�/ISS�14) restored �E2 splicing to the patternobserved for GE25� in COS-7 cells and to a lesser extent inPC12 cells. These experiments demonstrate that the ISS inhib-its use of a 5� splice site in a heterologous context, stronglysupporting the ISS linear model and that the ISS consists of 8nucleotides (positions 11–18).

ISS Function Is Position-independent—The effect of ISS po-sition on E10 splicing was investigated by inserting it at dif-ferent locations flanking the 5� splice site (Fig. 6A). When theISS was put into E10, a T nucleotide was added to the 8-nu-cleotide ISS so that the inserted sequence did not introduce aframeshift. When the ISS was 4 nucleotides upstream of the 5�splice site (E10-ISS, Fig. 6, A and B), E10 inclusion was virtu-ally abolished, presumably because of the presence of 2 func-tional ISS copies. Inactivation of the E10 ISS copy using theE10�14 mutation (E10-ISS�14) restored E10 splicing to al-

FIG. 3. Nucleotide substitutionswithin the ISM at I10 position 19. A,representative autoradiographs for COS-7and PC12 splicing assays. B, quantitationof E10 inclusion. Bar graphs are as de-scribed in Fig. 1. A corrected significancecriterion of p � 0.0125 was used. The sym-bols for p values are as in Figs. 1 and 2.

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most normal levels (compare hN and E10-ISS�14). Inactiva-tion of both ISS copies (construct E10-ISS�14/�14) resulted inincreased but not constitutive E10 inclusion relative to hN.Thus, the ISS maintains partial inhibitory function in spite ofmutation E10�14, as also observed in �E2 (see GE25�/ISS�14,Fig. 5D). When only the E10 ISS was present and the I10 ISSwas deleted (construct E10-ISS/�12–18), E10 inclusion wascomparable with the normal construct hN. Mutating the E10ISS (constructs E10-ISS�14/�12–18 and E10-ISS�14/�12–24)again resulted in elevated E10 inclusion at near constitutivelevels.

The ISS was also tested at locations more distant from the 5�splice site. Insertion into E10 at the �11 position resulted ininhibition of E10 inclusion in both COS-7 and PC12 cells, but

the effect was not altered by the E10�14 mutation (data notshown). The inhibition observed is probably because of thedisruption of a previously identified positive element at thislocation (21). Insertion of the ISS alone at I10 locations 31 and40, or both the ISS and ISM at I10 position 40 did not alter E10inclusion (data not shown). Thus, the ISS functions as a copynumber-dependent linear element that can function when inclose proximity to the 5� splice site.

The E10 3� Splice Site—Mammalian 3� splice sites have aninvariant AG at the 3� end of the intron, a polypyrimidine tract(PPT), and a BPS that includes the branch point A nucleotide15–50 nucleotides upstream of the exon (Fig. 7A). To see if thetau I9 PPT is suboptimal, the number of consecutive pyrimi-dines in the PPT was increased (�7T-11T and �3T-7T; Fig. 7,

FIG. 4. Substitutions in the 5� splicesite. A, proposed human E10 5� splice siteinteractions with U1 and U6 snRNAs. U1and U6 sequences are shown in italicsbelow and above the tau sequence, respec-tively. tau E10 and I10 sequences are rep-resented in uppercase and lowercase let-ters, respectively. Solid lines indicatepredicted base pair interactions betweenthe E10 5� splice site and snRNA se-quences. B, representative autoradio-graphs of splicing assays. C, quantitationof E10 inclusion. Bar graphs are as de-scribed in Fig. 1. A corrected significancecriterion of p � 0.01 was used. Signifi-cance levels for comparisons are as shownin Figs. 1 and 2.

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A and B). Both constructs showed constitutive E10 inclusion inCOS-7, PC12, and rat P1 neuronal cells, indicating that theE10 3� splice site is weak. This conclusion was confirmed byreplacing the 33-nucleotide tau I9 sequence in hN with a 42-nucleotide fragment containing the 3� splice site from human�-globin intron 1 (construct Glo3�ss). Near constitutive splicingoccurred in COS-7 and PC12 cells. The �-globin sequence wasused because it is a well studied splice site from a constitutiveexon with a strong BPS at �36 to �42 and a branch point A at�37 (33).

tau E10 has 2 candidate branch point A nucleotide at �23and �27 (Fig. 7A). To identify the tau BPS, both candidate Anucleotides were individually neutralized by changing each toa G (�23G and �27G, Fig. 7A). E10 inclusion was almostcompletely abolished in both constructs (Fig. 7B), and neither A

can compensate for the absence of the other. The strength ofthe BPS is determined primarily by its ability to hybridize tothe U2 snRNA sequence 5�-GUAGUA-3�. Both the candidateproximal and distal BPSs defined by �23A and �27A, respec-tively, appear weak with a 4/7 match against the degeneratemammalian consensus BPS 5�-UNCURAC-3� (where N is anynucleotide, R is a purine, and the branch point A is underlined).However, the proximal BPS may provide a stronger templatefor base pairing with U2 snRNA (Fig. 7A).

Utilization of the weak E10 5� splice site requires E10 en-hancer sequences (21). To ascertain whether use of the weakE10 3� splice site is also dependent on exon enhancer se-quences, mutants �3T-7T and �7T-11T were combined withdeletion E�5 in �3T-7T/E�5 and �7T-11T/E�5, respectively.Mutant E�5 (FTDP-17 mutation �280K) is an in-frame 3-nu-

FIG. 5. ISS function in a heterolo-gous construct. A, exon/intron junctionsequences for �E2 constructs. The �E2sequences are in uppercase letters and the�-globin intron 2 sequences in lowercaseletters. For cloning purposes, modifica-tions (arrows) were introduced into thenormal globin sequence in construct GE2,which destroy and create a BamHI (un-derlined) site in �E2 and �-globin intron2, respectively. Mutation at �E2 position�1 in GE25� is shown in italics, a substi-tution that weakens the �E2 5� splice sitesequence changing it from the normal�E2 sequence of 5�-AGgtgagt-3� to 5�-AT-gtgagt-3�, which is identical to the tauE10 5� splice site sequence. The insertedISS sequence in GE2/ISS is representedin italics and is located 11 nucleotidesdownstream of the �E2 5� splice site. Thec to t alteration (FTDP-17 mutationE10�14) at the fourth nucleotide in theISS is underlined in GE2/ISS�14 andGE25�/ISS�14. B, representative autora-diographs for splicing assays. Arrows in-dicate E2� and E2� fragments. Normaland cryptic E2� spliced products are rep-resented by a and a�, respectively. C,quantitation of splicing assays showingtotal �E2 levels in COS-7 and PC12 cells.The bar graphs are as described in Fig. 1.D, quantitation assays showing relativeuse of normal (white bar) versus cryptic(hatched bar) �E2 5� splice sites. A cor-rected significance criterion of p � 0.025was used for comparing constructs GE25�/ISS to GE25� for each cell line. Signifi-cance levels for comparisons are describedin Figs. 1 and 2.

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cleotide deletion within the PPE splicing enhancer element.This deletion results in almost complete inhibition of E10 in-clusion (11, 21). Strengthening the PPT immediately adjacentto the 3� AG could not compensate for the loss of the PPE inCOS-7 cells, as E10 splicing is abolished in mutant �3T-7T/E�5, whereas E10 splicing is maintained at normal levels inPC12 cells (Fig. 7B). When the PPT adjacent to the BPS isstrengthened, constitutive E10 splicing was observed in bothCOS-7 and PC12 cells despite the lack of PPE function inmutant �7T-11T/E�5. These results show that the PPE inter-acts with the weak 3� splice to control E10 inclusion.

Effects of Distal I9 and I10 Sequences on E10 Inclusion—Todetermine whether distal intron sequences regulate E10 inclu-sion, I9 and I10 sequences were extended from 33 and 51nucleotides (hN) to 567 and 190 nucleotides, respectively, inE10860 (Fig. 8A). In human I9, a tandem-repeat sequence is

located at nucleotides �21 to �55 and �69 to �116 upstreamof E10 (Fig. 8; Ref. 34). Mouse has only 1 repeat (34). ConstructhN contains 11 nucleotides of the 3� end of the repeat closest toE10 and includes both BPS with branch point A�s at �23 and�27. The distal I9 repeat present in construct E10860 alsocontains a sequence identical to the E10 BPS with the equiv-alent branch point A at position �81. There was no differencein E10 inclusion when hN and E10860 were compared. Thus,additional regulatory sequences were not apparent when ex-tended I9 and I10 sequences were tested, although the pres-ence of offsetting positive and negative regulatory elementscannot be excluded.

DISCUSSION

In MAPT, a complex hierarchy of enhancer and silencersequences in E10, I9, and I10 determines E10 alternative splic-

FIG. 6. ISS function within E10 sequences. A, tau sequence for the E10/I10 junction. E10 and I10 sequences are in uppercase and lowercaseletters, respectively. The normal ISS sequence is indicated by a filled oval and mutant ISS�14 by a hollow oval with a t in the center. ConstructE10-ISS contains a second ISS copy in E10, which replaces E10 sequences between �5 and �10 nucleotides upstream of the 5� splice site. Insertionof an in-frame normal or mutant ISS copy at the same position without replacing E10 sequences also show similar splicing effects (data not shown).In subsequent mutants, the ISS within E10, I10, or both was neutralized by a point mutation or deletion. Filled triangles indicate deletion of theISS (�12–18) or ISS and ISM (�12–24). Values to the right of the constructs represent the average percentage of inclusion of E10� transcripts fromthree transfection experiments with standard deviations (S.D.). A corrected significance criterion of p � 0.008 and p � 0.016 was used for COS-7and PC12 cells, respectively. Significance levels for each construct compared with normal E10 (hN) are given with symbols as in Fig. 1 and 2. B,representative autoradiographs of splicing assays.

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ing. Earlier work demonstrated that multiple ESE sequencesand an ESS in E10, a weak 5� splice site, and the ISS in I10regulate E10 inclusion (11, 21). Here, we identify an additionalI10 regulatory element, the ISM, and demonstrate that a weak3� splice site also contributes to E10 splicing regulation. TheISM interacts with the ISS, a weak 5� splice site, and E10regulatory elements to control E10 inclusion. Likewise, theweak 3� splice site interacts with cis-acting elements in E10.The ISS and ISM were defined in experiments using tran-siently transfected COS-7, PC12, and rat primary neurons,with most results being qualitatively and quantitatively simi-lar in all three cell types. Thus, the regulatory elements studiedhere are not neuron-specific and are generalizable to differentcell types.

The ISS and ISM form a novel 16-nucleotide bipartite regu-latory sequence immediately downstream of the weak E10 5�splice site. The ISS and ISM function antagonistically in reg-ulating E10 splicing, as they exert opposite effects on E10inclusion when individually inactivated (Fig. 2). Several linesof evidence indicate that the ISM and the ISS elements arediscrete. First, the ISS sequence alone, without the ISM, canfunction independent of position on either side of the 5� splicesite (Fig. 6). Second, the ISS can function in a heterologoussetting (Fig. 5). Third, the ISS inhibits E10 inclusion when theISM is inactivated by deletions �19–22 and �23–26 (Fig. 2) andby mutations (Fig. 3). In contrast, the ISM does not appear toact without the ISS, and functions to mitigate the ISS-medi-ated repression of E10 inclusion (Fig. 2).

FIG. 7. Characterization of the E10 3� splice site. A, human I9 sequence in hN. Shown are components of the 3� splice site that include theinvariant intronic 3�-AG dinucleotide, which is separated from an upstream BPS by a PPT. The BPS is usually located 15–50 nucleotides upstreamof the 3� cleavage site and is known to base pair with the U2 snRNA sequence AUGAUG. Arrows above the human I9 sequence indicate nucleotidesaltered in experimental constructs. Candidate branch point A nucleotides at positions �23 and �27 (underlined) were individually neutralized toa “g” in �23G and �27G, respectively. To strengthen the PPT, purine residues at positions �3, �4, and �7 as well as at positions �7, �10, and�11 were converted to “t” residues in mutants �3T-7T and �7T-11T, respectively. The box shows a comparison between the human BPS and theloosely conserved mammalian BPS consensus (N, any nucleotide; R, purine) and the U2 snRNA sequence. B, autoradiograph of RT-PCR productsshowing E10 inclusion.

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The close proximity of the ISM, ISS, and the 5� splice sitesuggests that these three elements interact. As shown previ-ously, ISS function requires that the adjacent 5� splice site besuboptimal (21). Here we show in E10 constructs that the ISScan function in a position-independent fashion as long as it isclose to the 5� splice site (Fig. 6), but will not function if thedistance to the 5� splice site too great. Thus trans-acting factorsthat bind to the ISS may directly hinder U1snRNP or othercomponents from correctly interacting with the 5� splice site,and therefore inhibit E10 inclusion. Results compatible withthis hypothesis were obtained when the ISS was inserteddownstream of a weak 5� splice site in �E2 globin constructs(Fig. 5). The ISS inhibited the use of the attenuated �E2 5� slicesite. Instead, an in-frame cryptic 5� splice site located 39 nu-cleotides downstream was utilized (Fig. 6). Interestingly, thiscryptic 5� splice site has a much weaker consensus sequencethan the attenuated �E2 5� splice site. The cryptic and weak-ened 5� splice sites show a 5/8 and a 7/8 match, respectively,with the mammalian 5� splice site consensus. Thus, the ISSinhibits use of a weak 5� splice site, thereby favoring splicing atthe next available cryptic site, even though the cryptic site hasa weaker consensus sequence.

The ISM does not appear to directly affect use of the 5� splicesite. Instead, the ISM has a positive effect on E10 splicing onlyin presence of the ISS. Thus, the ISM directly inhibits the ISSfunction of repressing use of the E10 5� splice site (Fig. 2). Aplausible mechanism for ISM function is that factors associat-ing with the ISM sterically hinder the stability or association ofISS-specific inhibitory factors, thus allowing indirect enhance-ment of E10 inclusion.3

The work presented here and previously (11, 21) is consistentwith ISS acting as a linear sequence rather than through asecondary RNA structure such as a stem loop (Fig. 1B). Thefollowing arguments support this conclusion. 1) The 8-nucleo-

tide ISS can function in a heterologous setting to inhibit use ofa weak 5� splice site (Fig. 5), a sequence context that does notform the proposed stem loop. 2) The ISS can be placed in E10near the 5� splice site and function to inhibit splicing (Fig. 6). 3)Although FTDP-17 mutations are proposed to disrupt the hy-pothetical stem loop, some (17) but not all compensatorychanges designed to restore stem loop base pairing act to re-store normal ISS function (11). 4) Double mutants containingI10 FTDP-17 mutations (E10�12, E10�13, E10�14, andE10�16) and a 3-nucleotide deletion in the E10 ESE (E�5; Ref.21) give variable E10 inclusion levels even though these I10mutations alone disrupt the stem loop to a comparable degree(21). 5) Both human and mouse E10/I10 junctions form stemloops of comparable stability, yet normal or chimeric humanand mouse constructs do not support a stem loop model for E10regulation either in transfected COS-7 cells (21) or in rat P1neurons.3 Additionally, preliminary UV-cross-linking experi-ments indicate that nuclear factors associate with normal butnot mutant ISS and ISM RNA sequences.3 Further work isnecessary to characterize these factors including their roles inregulating E10 splicing.

The ISS interaction with the 5� splice site components is notdominant. The antagonistic action of the ISS on 5� splice siterecognition and/or use can be over-ridden either by strength-ening an E10 ESE, or by strengthening the 5� splice site se-quence (21). Likewise, strengthening the 3� splice site alsooverrides ISS action (Fig. 7), demonstrating the complex inter-action between the different regulatory elements.

The E10 5� Splice Site—The tau E10 5� splice site is inher-ently weak as it deviates from the mammalian consensus basedon complementation with the 5� end of U1 snRNA (Fig. 4A).The predicted result is that U1 snRNA binds the tau E10 5�splice site weakly as recently confirmed by in vitro associationstudies (35). U1 snRNP, aided by other non-snRNP splicingfactors, associates transiently with the 5� splice site early dur-ing spliceosome assembly and serves to commit the pre-mRNA3 I. D’Souza and G. D. Schellenberg, unpublished observations.

FIG. 8. Effects of distal intron sequences on E10 inclusion. A, constructs hN and E10860 differ in the lengths of flanking human I9 andI10 sequences. Lengths in base pairs are below each construct. Striped boxes are tandem repeats in I9 located upstream of E10 (34). The stripedbox in hN represents the last 11 nucleotides of the 35-nucleotide proximal tandem repeat that is present in I9. B, representative autoradiographsof E10 splicing assays.

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to the splicing pathway (36). Although this interaction alonedoes not determine the position or even use of the 5� splice site,it ensures at an early step proper fidelity in 5� splice siteselection (31). More importantly, the 5� splice site interactionwith U1 snRNP is replaced by subsequent interactions with U5and U6 snRNPs in a process that defines the final position ofthe 5� splice site. An invariant sequence ACAGAG within U6snRNA interacts with intron positions 5–10 downstream of the5� cleavage site (Fig. 4A; Ref. 37). For tau, previous studiesshowed that strengthening the E10 5� splice site either byincorporating FTDP-17 mutations S305N or E10�3 (9, 11, 18)or replacing it with a constitutive 5� splice site from human�-globin exon 2 (21) caused E10 to oversplice. Here, resultsfrom �7–10 and substitution analyses in I10 just downstreamof the conserved 5� splice site sequence (Figs. 2B and 4C)indicate that nonconserved I10 sequences from positions 7 upto 10 are part of the weak 5� splice site sequence. Thus, aninteresting feature regarding E10 splicing regulation is thatboth U1 and U6 snRNPs are predicted to interact weakly withthe normal E10 5� splice site region, as substitutions within I10positions 8 and 10 that are thought to strengthen U1 snRNPand U6 snRNP interactions increase E10 inclusion.

Within the pre-mRNA/spliceosome complex, U6 snRNA isthought to possess the catalytic activity for cleaving 5� and 3�splice sites (38). Although our work predicts a weak interactionbetween the E10 5� splice site and U6 snRNA, not all substi-tutions that are predicted to increase U6 snRNA associationwith the 5� splice site result in increased E10 splicing (t(�10)gin COS-7 cells; Fig. 4C). One explanation is that this particularsubstitution may alter the association of other splicing factorswithin the I10 region. For example, interaction of the yeast U1snRNP protein Nam8p with intron positions 6–13 is requiredfor efficient 5� splice site recognition by U1 snRNP, especiallywhen the 5� splice site is weakened (26). Several other snRNP-specific and non-snRNP splicing factors have also been cross-linked to the 5� splice site (27). Splicing is most enhanced indouble mutant c(�8)t/t(�10)g in both COS-7 and PC12 cells,suggesting possible cooperative effects when both U1 and U6

snRNA associations with a weak 5� splice site are strength-ened. The important contributions of other cis-acting splicingsequences on E10 5� splice site use must also be taken intoaccount as seen by mutations or substitutions elsewhere inE10, I9, or I10 sequences that dramatically affect the outcomeof E10 splicing (21). These splicing regulatory sequences inassociation with trans-acting splicing factors help in stabilizing(or destabilizing) spliceosome interactions with the pre-mRNAtemplate.

The E10 3� Splice Site Is Regulatory—The 3� splice site isweak, as shown when the PPT is strengthened by increasingthe number of consecutive pyrimidines or where the entire I9sequence is replaced with a constitutive globin 3� splice site(Fig. 7B). Most enhancer-dependent introns are associatedwith a weak or suboptimal PPT (Ref. 39; reviewed in Ref. 40),which when strengthened overcomes the need for an enhancer.ESEs not only aid in the recognition of weak splice sites (re-viewed in Ref. 41), but some also function in overcoming theeffects of splicing silencers (42). This is certainly true for mu-tant �3T-7T (compare �3T-7T to double mutant �3T-7T/E�5,Fig. 7B). Strengthening the PPT adjacent to the non-consensusBPSs in �7T-11T allows for efficient recognition of the 3� splicesite independent of enhancer and silencer sequences (compare�7T-11T and �7T-11T/E�5, Fig. 7B). Early in the splicingpathway, splicing factors such as the 35-kDa U2 auxiliaryfactor (U2AF35) associates with the 3� AG, which in turn sta-bilizes the interaction of U2AF65 with the PPT. U2AF65 in turnstrengthens the recruitment of U2 snRNP to the BPS. Theseinteractions are critical in the case of weak 3� splice sites,where SR proteins in association with ESEs increase the affin-ity of U2AF and U2 snRNP for the upstream 3� splice site. Thepresence of splicing silencer sequences further complicatesthese interactions. The PPT in �7T-11T may provide a stron-ger template for U2AF65 interaction than in �3T-7T. Although3 purines are converted to pyrimidines (all uridines) in both�3T-7T and �7T-11T, they do differ in the number of consec-utive pyrimidines. Construct �7T-11T has 17 continuous pyri-midines adjacent to the BPS, whereas �3T-7T has a 10- and a

FIG. 9. Model for tau E10 splicing regulation. Shown are putative splicing factor interactions with exonic and intronic splicing regulatorysequences that modulate use of the weak E10 5� and 3� splice sites. E10 splicing elements include three ESE elements (SC35-like, PPE, and ACE)at the 5� end of E10 and two ESE elements at the 3� end of E10, as well as an 18-nucleotide ESS sequence between these two ESEs. I10 regulatorysequences include the bipartite regulatory ISS and ISM element. SR factors usually in association with ESE elements recruit and stabilize U1 andU2 snRNP interactions with the 5� and 3� splice sites, respectively. These exon-bridging interactions ultimately help in defining the exon. For tauE10 definition, the positive roles of the ESEs (blue arrows with �) and ISM are critical because of weak 3� and 5� splice sites as well as theinhibitory ESS and ISS elements (red arrows with �). The positive role of the ISM on E10 splicing is indirect and ISS-dependent. Known diseasemutations are shown at the bottom.

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7-nucleotide pyrimidine stretch interrupted by two guanidines.The difference in �3T-7/E�5 splicing between COS-7 and PC12cells (Fig. 7) may be a result of the fact that PC12 are neuronalcells and COS-7 are not. The difference could also be caused bythe recently identified splicing factor U2AF26 that is particu-larly enriched in brain and shares a similar function withU2AF35 in recruiting U2AF65 to weak 3� splice sites (43). Thus,the E10 PPE is required not only for recruitment of splicingfactors to the weak 3� splice site, but also needs to overcome theeffects of splicing silencer sequences within E10 and I10. E10inclusion also involves the use of at least two overlapping andweak BPSs with branch point A nucleotides at nucleotides �23and �27 upstream of E10. In addition to the weak PPT, mul-tiple weak BPSs also contribute to the weak 3� splice site inregulating E10 splicing.

Model for Regulation of tau E10 Splicing—A model for tauE10 splicing regulation is proposed (Fig. 9), where multipleweak interactions between factors binding to sequences in E10,I9, and I10 ultimately help in defining E10. Rather than anysingle sequence, multiple enhancer and silencer sequencesmodulate use of the weak 5� and 3� splice sites that border E10.In both constitutively and alternatively spliced exons, SR pro-teins interact with ESE elements and stabilize interactionsbetween the splicing machinery (snRNPs) and splice sitesacross the length of the exon, thus defining the exon (44). Forexample, ESE-bound SR factors SC35 and ASF/SF2 interactdirectly with U1 snRNP-specific factor U1 70K and stabilize U1snRNP association with the 5� splice site (36, 39, 45). At the 3�splice site region, the U2AF65 and U2AF35 subunits of theU2AF heterodimer interact with the PPT and 3�-AG, respec-tively. These 3� splice site associations are stabilized throughESE-associated SR factors that directly interact with U2AF35.U2AF65 in turn stabilizes U2 snRNP interaction with the BPS.The involvement of ESEs is particularly important when eithersplice site is weak. In the case of tau E10, both splice sites areweak, and thus multiple ESE sequences are required for E10inclusion (21). In addition to their role in exon definition andsplice site selection, SR and other proteins may also antagonizesplicing inhibitory factors in association with splicing silencersequences, such as the ESS and ISS sequences in tau E10splicing. An additional level of complexity is presented by thejuxtaposition of the 5� splice site with the ISS and ISM ele-ments in I10.

The proposed interactions of ESE and ESS sequences withweak 3� and 5� splice sites for MAPT E10 are consistent withthe exon definition model for splicing (44). According to thismodel, components of the splicing machinery in associationwith the 5� and 3� splice sites communicate across the exon todefine the exon/intron boundary. This interaction across theexon is mediated and strengthened by SR proteins bound toenhancer sequences in the exon. Weakened interactions withsplice sites at either end of the exon allow exon skipping.

E10 Regulation and FTDP-17—Work in cell culture modelsusing a number of different cell lines has demonstrated thatE10 is regulated by at least eight cis-acting elements in thecontext of weak 3� and 5� splice sites. Evidence supporting thefunction of these elements in vivo comes from the identificationof FTDP-17 mutations in six of the cis-acting sequences (Fig. 9).Mutations include intronic mutations in the ISS (7, 9, 16), amissense mutation (N279K) and a deletion mutation (�280K)in the PPE (11, 16, 19), a silent mutation (L284L) in the ACE(11), a deletion mutation (�296N; Ref. 46), a silent mutation(N296N; Ref. 47) and a missense mutation (N296H, Ref. 48) inthe ESS, and a new mutation E10�192 in the ISM. FTDP-17mutations S305N, S305S, and E10�3, which are in the 5� splice

site consensus sequence, provide in vivo evidence that the 5�splice site is functionally suboptimal. The fact that these splic-ing mutations cause severe neurodegenerative disease demon-strates the functional significance of these regulatory elements.

Acknowledgments—We thank Leojean Anderson and Elaine Loomisfor technical assistance and Patrick A. Navas for PAC P�(BglII). Wealso thank Soren Impey and Daniel R. Storm for preparation andtransfection of rat P1 neuron cultures.

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Ian D'Souza and Gerard D. Schellenberg Splice Sites ′ and 3′Weak 5

Exon 10 Expression Involves a Bipartite Intron 10 Regulatory Sequence andtau

doi: 10.1074/jbc.M203794200 originally published online May 8, 20022002, 277:26587-26599.J. Biol. Chem. 

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